Comprehensive profiling of DNA methylation in colorectal cancer reveals subgroups with distinct clinicopathological and molecular features

Abstract

Background: Most previous studies of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) have been conducted on a relatively small numbers of CpG sites. In the present study we performed comprehensive DNA methylation profiling of CRC with the aim of characterizing CIMP subgroups.

Methods: DNA methylation at 1,505 CpG sites in 807 cancer-related genes was evaluated using the Illumina GoldenGate methylation array in 28 normal colonic mucosa and 91 consecutive CRC samples. Methylation data was analyzed using unsupervised hierarchical clustering. CIMP subgroups were compared for various clinicopathological and molecular features including patient age, tumor site, microsatellite instability (MSI), methylation at a consensus panel of CpG islands and mutations in BRAF and KRAS.

Results: A total of 202 CpG sites were differentially methylated between tumor and normal tissue. Unsupervised hierarchical clustering of methylation data from these sites revealed the existence of three CRC subgroups referred to as CIMP-low (CIMP-L, 21% of cases), CIMP-mid (CIMP-M, 14%) and CIMP-high (CIMP-H, 65%). In comparison to CIMP-L tumors, CIMP-H tumors were more often located in the proximal colon and showed more frequent mutation of KRAS and BRAF (P<0.001).

Conclusions: Comprehensive DNA methylation profiling identified three CRC subgroups with distinctive clinicopathological and molecular features. This study suggests that both KRAS and BRAF mutations are involved with the CIMP-H pathway of CRC rather than with distinct CIMP subgroups.

Figure 1

Unsupervised hierarchical clustering of 202 tumor-specific probes (rows) in 91 CRC (columns). The 3 tumor clusters generated by this analysis were termed CIMP-High (CIMP-H), CIMP-Mid (CIMP-M) and CIMP-Low (CIMP-L). Clinicopathological and molecular features are shown above the heatmap. White rectangles are cases with missing data. Gender: female (red), male (blue); Age: ≥ 67 years (red), < 67 (blue); Tumor location: proximal (red), distal (blue); Tumor stage (ACPS): A or B (blue), C or D (red); Lymphovascular invasion (LVI): present (red), absent (blue); Extramural vascular invasion (EMVI): present (red), absent (blue); Perineural invasion (PNI): present (red), absent (blue); Tumor infiltrating lymphocytes (TILS): present (red), absent (blue); CIMP^W: CIMP^W-high (red), CIMP^W-low (yellow), CIMP^W-negative (blue); BRAF: mutant (red), wildtype (blue); KRAS: mutant (red), wildtype (blue); Microsatellite instability (MSI): positive (red), negative (blue). Five CpG clusters (A-E) were apparent from the analysis and showed differential methylation amongst the 3 CIMP subgroups.